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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

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Materials

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Medicine

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Published: August 11th, 2023

DOI:

10.3791/65890

1Department of Oral Pathology, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, 2Research Unit of Precision Pathologic Diagnosis in Tumors of the Oral and Maxillofacial Regions, Chinese Academy of Medical Sciences (2019RU034), 3Central Laboratory, Peking University School and Hospital of Stomatology

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Oral submucous fibrosis (OSF) is a chronic, insidious disease that develops mainly in the buccal mucosa and results in restricted mouth opening1. While OSF is a multifactorial disease, areca nut or betel nut chewing is the main cause of OSF2,3. Because of this geographically specific habit, OSF is predominantly concentrated in populations in Southeast and South Asia3. The common histological features of OSF include abnormal collagen deposition in the connective tissue beneath the oral mucosal epithelium, vascular stenosis, and occlusion1. ....

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This study was approved by the institutional review board of Peking University School and Hospital. Informed consent was obtained from the patients. The tissue samples used in this study were deidentified. The study scheme is shown in Figure 1.

1. Sample preparation

  1. Cut formalin-fixed paraffin-embedded oral submucous fibrosis tissues into continuous sections of 3 µm and 10 µm thickness on a microtome.
  2. Unfold the sec.......

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By performing laser microdissection of OSF tissues, we captured samples of dysplastic epithelium, stroma beneath the dysplastic epithelium, atrophic epithelium, and stroma beneath atrophic epithelial tissue (Figure 1).Through extracting DNA and low-depth whole genome sequencing, we were able to analyze morphology-related copy number alterations (CNA)15. CNA is a common form of genomic instability associated with an increased risk of malignant transformation in OPMD

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This protocol reported a pipeline to capture OSF tissue samples with morphological and spatial information for further spatial-omic analyses through laser microdissection. From the representative results, we identified different CNA patterns among various morphology-related samples.

OSF, a type of OPMD, is a common precancerous condition of oral squamous cell carcinoma6. Genomic instability has been reported to be associated with the development and malignant transforma.......

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This work was supported by research grants from the National Nature Science Foundation of China (81671006, 81300894), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-038), National clinical key discipline construction project (PKUSSNKP-202102), Innovation Fund for Outstanding Doctoral Candidates of Peking University Health Science Center (BMU2022BSS001).

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NameCompanyCatalog NumberComments
Adhesion microscope slidesCITOTESTREF.188105
Div-haematoxylinYiLi20230326
Eosin solutionBASOBA4098
EthanolPEKING REAGENTNo.32061
Harris hematoxylin dye solutionYiLi20230326
Hot plateLEICAHI1220
Laser capture microdissection systemLEICALMD7Machine
Laser microdissection microsystemLEICA8.2.3.7603Software
Micromount mounting mediumLEICAREF.3801731
Microscope cover glassCITOTESTREF.10212450C
MicrotomeLEICARM2235
PCR tubesAXYGEN16421959
PEN-membrane slidesLEICANo.11505158
Re-blue solutionYiLi20230326
Ultrapure distilled waterInvitrogenREF.10977-015
XylenePEKING REAGENTNo.33535

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